Treatment of skin, and wound repair, with thymosin beta 4

ABSTRACT

Compositions and methods for treatment of skin utilizing thymosin β4.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 09/772,445, filedJan. 29, 2001, which is a continuation of the PCT/US99/17282, filed Jul.29, 1999, which claims benefit of U.S. Provisional Application Ser. No.60/094,690, filed Jul. 30, 1998. The previously mentioned applicationsare explicitly incorporated herein by reference in their entirety forall purposes.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made in part with funds from the National Institutesof Health, Intramural Program. The government may have certain rights inthe invention.

TECHNICAL FIELD OF THE INVENTION

The present invention relates generally to tissue repair and morespecifically to methods of wound healing using thymosin B4.

BACKGROUND OF THE INVENTION

Inadequate methods and compositions to effectively heal chronic woundsis a significant health care problem. Impaired wound healing increasesthe chances of mortality and morbidity. This problem is especiallyprominent in patients with diabetes who develop severe, life threateningwounds on body extremities. Chronic diabetic foot ulcers often lead toamputations. These wounds are often the result of poor circulationderived from the diabetic patients' insulin-compromised cells as well asimpaired vascularization of the wound bed, reduced infiltration of germfighting cells and reduced tissue epithelialization. As a result, mostcurrent therapies include attempts to revascularize the wound bed andprevent infection.

Wounds in non-compromised tissues undergo a complex and ordered seriesof events to repair the tissue. The series of events may includeinfiltration of immune cells as part of the process to remove anddestroy necrotic tissue, increased vascularization by angiogenic factorsand increased cell proliferation and extracellular matrix deposition.Although the basic process of tissue repair has been characterized, theindividual steps and factors necessary to carry out this complex seriesof events are not well understood. The identification of individualsteps and factors could lead to improved methods for the treatment ofdiseases resulting from inadequate wound repair processes.

Previous studies have used the “scratch” wound closure assay to assessthe potential effects of an agent on in vitro cell migration. Thoughinformative, such a test does not mimic the dynamic in vivo woundhealing conditions to the extent that not all factors involved in woundclosure are present in the in vitro assay. For this reason, in vivasystems have been developed to assess the ability of an agent or factorto modulate wound healing activities.

Using these types of in vitro models, a number of specific growthfactors have been recognized for their effect on angiogenesis. One suchgrowth factor is TGF-β. This family of dimeric proteins includes TGF-β1,TGF-β2, TGF-β3, TGF-β4, and TGF-β5 which regulate the growth anddifferentiation of many cell types. This family of proteins exhibits arange of biological effects from stimulating the growth of some celltypes (Noda et al., (1989) Endocrinology, 124:2991-2995) and inhibitingthe growth of other cell types (Goey et al., (1989) J. Immunol.,143:877-880; Pietenpol et al., (1990) Proc. Nat'l. Acad. Sci. USA,87:3758-3762). TGF-β has also been shown to increase the expression ofextracellular matrix proteins, including collagen and fibronectin(Ignotz et al., (1986) J. Biol. Chem., 261:4337-4345) and acceleratesthe healing of wounds (Mustoe et al., (1987) Science, 237:1333-1335).

Another growth factor recognized for its effect on angiogenesis isPlatelet Derived Growth Factor (PDGF). PDGF was originally found to be apotent mitogen for mesenchymal derived cells (Ross R. et al. (1974) ProcNat'l Acad Sci USA 71(4):1207-1210.; Kohler N. et al. (1974) Exp. CellRes. 87:297-301). Further studies have shown that PDGF increases therate of cellularity and granulation in tissue formation. Wounds treatedwith PDGF have the appearance of an early stage inflammatory response,including an increase in neutrophils and macrophage cell types at thewound site. These wounds also show enhanced fibroblast function (Pierce,G F et al. (1988) J. Exp. Med. 167:974-987). Both PDGF and TGFβ havebeen shown to increase collagen formation, DNA content, and proteinlevels in animal studies. (Grotendorst, G R et al. (1985) J. Clin.Invest. 76:2323-2329.; Sporn, M B et al. (1983) Science 219:1329). Theeffect of PDGF in wound healing has been shown to be effective in humanwounds. In human wounds, PDGF-AA expression is increased within pressureulcers undergoing healing. The increase of PDGF-AA corresponds to anincrease in activated fibroblasts, extracellular matrix deposition, andactive vascularization of the wound. Furthermore, such an increase inPDGF-AA is not seen in chronic non-healing wounds. A number of othergrowth factors having the ability to induce angiogenesis and woundhealing include, Vascular Endothelial Growth Factor (VEGF), KeratinocyteGrowth Factor (KGF) and basic Fibroblast Growth Factor (bFGF).

However, most of these growth and angiogenic factors have side effects.Accordingly, there is a need for additional factors useful in promotingwound repair.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that thymosin β4 (Tβ4)accelerates wound healing and stimulates wound repair. Based on thisfinding, it is now possible to develop methods for accelerating woundhealing in subjects having wounds in need of such treatment.

In a first embodiment, the invention provides a method for promotingwound repair in a subject in need of such treatment by administering tothe subject or contacting the site of the wound with a wound-healingeffective amount of a composition containing a wound healing polypeptidecomprising the amino acid sequence LKKTET (SEQ ID NO:1) and conservativevariants thereof having wound healing activity. In one aspect of themethod, the wound healing polypeptide is Tβ4 or an isoform of Tβ4.

In another embodiment, the invention provides a method for promotingtissue repair in a tissue in need of such treatment by contacting thetissue with an effective amount of a composition containing a woundhealing polypeptide comprising the amino acid sequence LKKTET (SEQ IDNO:1) and conservative variants thereof having wound healing activity,or nucleic acid encoding a wound healing polypeptide. In one aspect ofthe method, a wound healing peptide is Tβ4 or an isoform of Tβ4. Thetissue may be contacted either in vivo or ex vivo.

In yet another embodiment, the invention provides a method of modulatingwound repair in a subject in need of such treatment by systemic deliveryof a wound-healing effective amount of a wound healing polypeptidecomprising the amino acid sequence LKKTET (SEQ ID NO:1) and conservativevariants thereof having wound healing activity. In one aspect of themethod, a wound healing peptide is Tβ4 or an isoform of Tβ4.

In yet another embodiment, the present invention provides a method forstimulating epithelial cell migration at the site of a wound bycontacting the wound with an effective amount of a Tβ4 polypeptide.

In another embodiment, the invention provides a method of diagnosing apathological condition in a subject characterized by a wound healingdisorder associated with Tβ4, including obtaining a sample suspected ofcontaining Tβ4 from the subject, detecting a level of Tβ4 in the sampleand comparing the level of Tβ4 with the level found in a normal sample(i.e., a standard sample).

In another embodiment, the invention provides a method of ameliorating awound healing disorder associated with Tβ4, including treating a subjecthaving the disorder with a composition which modulates Tβ4 activity orthe activity of a Tβ4 isoform.

In yet another embodiment, the present invention provides pharmaceuticalcompositions comprising a wound healing polypeptide comprising the aminoacid sequence LKKTET [SEQ ID NO:1] (SEQ ID NO:1) and conservativevariants thereof having wound healing activity and a pharmaceuticallyacceptable carrier. In one aspect, the wound healing polypeptide is Tβ4or an isoform of Tβ4.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic drawing of a wound.

FIG. 2 is a bar graph which shows the effect of topical and systemicdelivery of Tβ4 on the width of a punch wound as compared to control.(A) Topical delivery of 5 μg/50 μl was performed on three of the sixwounds in each animal on the day of wounding and at 48 hours afterwounding. (B) Intraperitoneal injections of 60 μg/300 μl were done onthe day of the wounding and thereafter every other day. Control animalswere treated similarly with saline. Measurements are expressed as themean percent decrease±SEM.

FIG. 3 is a bar graph which shows the effect of topical and systemicdelivery of Tβ4 on the gap of a punch wound as compared to control. (A)Topical delivery of 5 μg/50 μl was performed on the day of wounding andat 48 hours after wounding. (B) Intraperitoneal injections of 60 μg/300μl were done on the day of the wounding and thereafter every other day.Measurements are expressed as the mean percent decrease±SEM.

FIG. 4 is a histological section, stained with H&E, demonstrating theappearance of control and thymosin β4 treated wounds at lowmagnification and higher magnification. Wounds are from day 7 asdescribed in the legend to FIG. 2. Arrows indicate the edges of theoriginal wound. (A) Control wound treated with saline. Migration of theepithelium is visible at the wound edges and debris are visible over theunhealed wound. (B) Increased re-epithelialization of the wound occurredwhen Tβ4 was injected intraperitoneally (60 μg/300 μl on alternatedays). (C) Topical treatment (5 μg/50 μl of Tβ4) resulted in completereepithelialization of the wound epidermis. Boxed areas are the locationof the higher magnification fields (D-F). (D-F) Dermis near dermal andepidermal junction. (D) Control showing few cells near the dermis andlittle neovascularization. (E) and (F) Dermis showing granulation tissueinfiltrated with fibroblasts and extensive neovascularization(arrowheads). (E) Intraperitoneal treatment and (F) topical applicationboth resulted in significant new capillaries. (Scale bar=1 mm).

FIG. 5 shows histological sections of 7 day wounds showing collagendeposition/accumulation. Masson's trichrome staining shows collagen andendothelial cells. (A) Low magnification view of a control wound treatedwith saline. (B) and (C). Low magnification views of wounds where Tβ4was injected intraperitoneally (B) or applied topically (A). Boxed areasare the location of the higher magnification fields (D-F). Arrowsindicate the edges of the original wound. (D) Control wound at highermagnification showing baseline collagen accumulation. Treatmentintraperitoneally (E) or (F) topically resulted in enhanced collagenproduction/accumulation compared to wounds treated with saline. (Scalebar=1 mm).

FIG. 6 shows Tβ4 stimulated keratinocyte migration in Boyden chamberassays. (A) Tβ4 in the lower wells of the chamber resulted in a 2-3 foldincrease in migration on filters coated with collagen IV. The positivecontrol, conditioned media, also showed increased migration over mediaalone.

FIG. 7 shows a graph demonstrating the migration of corneal epithelialcells at various concentrations of Tβ4.

FIG. 8 shows a graph representing corneal re-epithelialization in ratcorneas in the presence and absence of Tβ4.

FIG. 9 shows a graph representing corneal re-epithelialization in thepresence and absence of various concentrations of Tβ4.

FIG. 10 shows an amino acid sequence of Tβ4 (SEQ ID NO:3).

FIG. 11 a shows the amino acid sequence of several known isoforms ofTβ4, and FIG. 11 b their phylogenetic distribution (Tβ₄:SEQ ID NO:3, Tβ₄^(Ala):SEQ ID NO:4, Tβ₄ ^(Xen):SEQ ID NO:5, Tβ₉:SEQ ID NO:6, Tβ₉^(Met):SEQ ID NO: 7, Tβ₁₀:SEQ ID NO:8, Tβ₁₁:SEQ ID NO:9, Tβ₁₂:SEQ IDNO:10, Tβ₁₂ ^(Perch):SEQ ID NO:11, Tβ₁₃:SEQ ID NO:12, Tβ₁₄:SEQ ID NO:13,Tβ₁₅:SEQ ID NO:14, Tβ^(Scallop):SEQ ID NO:15 Tβ^(sea urchin): SEQ IDNO:16). N-terminal acetylation is indicated by “ac.” Residues between 13and 24 are thought to be important for actin binding.

DETAILED DESCRIPTION OF THE INVENTION

Thymosin β4 was initially identified as a protein that is up regulatedduring endothelial cell migration and differentiation in vitro. Thymosinβ4 was originally isolated from the thymus and is a 43 amino acid, 4.9kDa ubiquitous polypeptide identified in a variety of tissues. Severalroles have been ascribed to this protein including a role in endothelialcell differentiation and migration, T cell differentiation, actinsequestration and vascularization. One biological activity of thymosinβ4 (Tβ4), as shown herein, effects tissue repair and wound healing.Another activity of Tβ4 is anti-inflammatory activity.

The present invention resulted from investigation of the effects of Tβ4on wound healing. In vivo results have demonstrated that topical andsystemic delivery of Tβ4 promotes wound healing. Additional experimentsdemonstrated that Tβ4-treated wounds have increased extracellular matrixdeposition in the wound bed.

The present invention identifies Tβ4 as an active factor in promotingwound closure and tissue repair in vivo as well as increasing epithelialcell migration. In vivo administration of Tβ4 indicates that cellmigration, angiogenesis and extracellular matrix deposition arestimulated at or above the levels observed for migration, angiogenesisand matrix deposition in control animals. Tβ4 promotes wound closurewhen administered systemically (e.g., intra-peritoneally) and topicallyin wounded animal models. Increased levels of collagen were alsoobserved in treated wounds showing that Tβ4 treatment can alsoaccelerate wound contraction and stimulate the healing process.

The methods of the invention result from the identification of theeffect of Tβ4 on wound healing. In vivo, Tβ4 stimulates wound healing ina fill thickness punch wound (see Example 1) and in repair ofeye-related wounds (Example 4). When given either topically orsystemically (e.g., intra-peritoneally) Tβ4 accelerated closure andhealing of wounds (see Example 1, 4, and 5).

Promoting Tissue Regeneration

In one embodiment, the invention provides a method for acceleratingwound healing in a subject by contacting a wound with a wound-healingeffective amount of a composition which contains Tβ4 or a Tβ4 isoform.The contacting may be topically or systemically. Examples of topicaladministration include, for example, contacting the wound with a lotion,salve, gel, cream, paste, spray, suspension, dispersion, hydrogel,ointment, or oil comprising Tβ4. Systemic administration includes, forexample, intravenous, intraperitoneal, intramuscular injections of acomposition containing Tβ4 or a Tβ4 isoform. A subject may be anymammal, preferably human.

In addition, Tβ4 or a Tβ4 isoform is therapeutically valuable in caseswhere there is an impaired wound healing process, such as in woundhealing compromised subjects. By “wound healing compromised” is meantsubjects which have a reduced, decreased, or inability to recover from awounding or trauma, due to recurrent wounding, trauma or inability ofthe subject's natural system to properly effectuate wound healing. Forexample, steroids reduce the ability of a subject to heal as compared toa subject which is not on steroids. Other such wounds present incompromised subjects include, but are not limited to, skin wounds suchas diabetic ulcers, venus ulcers or pressure ulcers. Additionally, Tβ4or a Tβ4 isoform is therapeutically valuable to augment the normalhealing process.

As used herein, a “wound-healing effective amount” of a compositioncontaining Tβ4 or a Tβ4 isoform for use in wound healing is defined asthat amount that is effective in promoting tissue regeneration andrepair. The “wound-healing effective amount” may be the therapeuticallyeffective amount. Diseases, disorders or ailments possibly modulated byTβ4 or a Tβ4 isoform include tissue repair subsequent to traumaticinjuries or conditions including arthritis, osteoporosis and othermusculo-skeletal disorders, burns, ulcers and other skin lesions,neurological and nerve disease and cardiovascular diseases includingischemia and atherosclerosis. Other potential tissues which can betreated by the methods and compositions of the invention includeepidermal, eye, uro-genital, gastrointestinal, cardiovascular, muscle,connective, and neural tissues. The term “induce”, “induction” or“effect” as used herein, refers to the activation, stimulation,enhancement, initiation and/or maintenance of cellular mechanisms orprocesses necessary for the formation of a tissue or a portion thereof,repair process or tissue development as described herein.

Modulation of Wound Healing

Wound healing, tissue regeneration and tissue repair result from acomplex process that includes the proliferation and migration ofinflammatory cells, endothelial cells, stromal cells and parenchymalcell, the deposition of extracellular matrix materials and the growth ofnew blood vessels, particularly capillaries. This complex process playsa crucial role in such beneficial functions as embryogenesis, the femalereproductive cycle, as well as such abnormal functions as arthritis,chronic ulcerations and neuro-degenerative diseases.

In another embodiment, the invention provides a method for modulatingwound healing in a subject or a tissue including contacting the subjector tissue with an effective wound-healing amount of a compositioncontaining Tβ4 or a Tβ4 isoform. It is envisioned that Tβ4 or a Tβ4isoform can be administered topically or systemically to prevent ortreat a damaged tissue including, for example, tissues damaged due toischemia, including ischemic brain, bone and heart disease, damage tocorneal or retinal tissue of the eye, and damage to epithelial tissue,including skin.

In addition, the method of the invention is useful in promoting woundhealing in tissues by promoting angiogenesis in tissue deprived ofadequate blood flow. For example, a composition containing Tβ4 canpromote the healing of chronic ulcers by increasing blood supply to thetissue site as well as increasing keratinocyte migration to close awound.

Tβ4 isoforms have been identified and have about 70%, or about 75%, orabout 80% or more homology to the amino acid sequence of Tβ4 set forthin FIG. 10 (SEQ ID NO:3). Such isoforms include, for example, Tβ4^(ala),Tβ9, Tβ10, Tβ11, Tβ12, Tβ13, Tβ14 and Tβ15 (FIG. 11; see also, Mihelićet al., (1994) Amino Acids, 6:1-13, which describes the amino acidsequence of other Tβ4 isoforms, and is incorporated herein byreference).

Thus, it is specifically contemplated that known Tβ4 isoforms, such asTβ4^(ala), Tβ9, Tβ10, Tβ11, Tβ12, Tβ13, Tβ14, and Tβ15, as well as Tβ4isoforms not yet identified, will be useful in the methods of theinvention. As such Tβ4 isoforms are useful in the methods of theinvention, including the methods practiced in a subject, the inventiontherefore further provides pharmaceutical compositions comprising Tβ4isoforms Tβ4^(ala), Tβ9, Tβ10, Tβ11, Tβ12, Tβ13, Tβ14, and Tβ15 and apharmaceutically acceptable carrier.

In addition, other proteins having actin sequestering or bindingcapability, or that can mobilize actin or modulate actin polymerization,as demonstrated in an appropriate sequestering, binding, mobilization orpolymerization assay, or identified by the presence of an amino acidsequence that mediates actin binding, such as LKKTET (SEQ ID NO:1), forexample, can similarly be employed in the methods of the invention. Suchproteins including gelsolin, vitamin D binding protein (DBP), profilin,cofilin, depactin, Dnasel, vilin, fragmin, severin, capping protein,-actinin and acumentin, for example. As such methods include thosepracticed in a subject, the invention further provides pharmaceuticalcompositions comprising gelsolin, vitamin D binding protein (DBP),profilin, cofilin, depactin, Dnasel, vilin, fragmin, severin, cappingprotein, -actinin and acumentin as set forth herein. Thus, the inventionincludes the use of wound healing polypeptide comprising the amino acidsequence LKKTET (SEQ ID NO:1) and conservative variants thereof.

As used herein, the term “conservative variant” or grammaticalvariations thereof denotes the replacement of an amino acid residue byanother, biologically similar residue. Examples of conservativevariations include the replacement of a hydrophobic residue such asisoleucine, valine, leucine or methionine for ariother, the replacementof a polar residue for another, such as the substitution of arginine forlysine, glutamic for aspartic acids, or glutamine for asparagine, andthe like.

Tβ4 has been localized to a number of tissue and cell types and thus,agents which stimulate the production of Tβ4 can be added to acomposition to effect Tβ4 production from a tissue and/or a cell. Agentsthat effect wound repair can also be included in such a composition toaugment the wound healing process. Such agents include members of thefamily of growth factors, such as insulin-like growth factor (IGF-1),platelet derived growth factor (PDGF), epidermal growth factor (EGF),transforming growth factor beta (TGF-β), basic fibroblast growth factor(bFGF), thymosin α1 (Tα1) and vascular endothelial growth factor (VEGF).More preferably, the agent is transforming growth factor beta (TGF-β) orother members of the TGF-β superfamily. Tβ4 compositions of theinvention aid in wound healing by effectuating growth of the connectivetissue through extracellular matrix deposition, cellular migration andvascularization of the wound bed.

Additionally, agents that assist or stimulate the wound healing processmay be added to a composition along with Tβ4 or a Tβ4 isoform to furthermodulate the wound healing process. Such agents include angiogenicagents, growth factors, agents that direct differentiation of cells,agents that promote migration of cells and agents that stimulate theprovision of extracellular matrix materials in the wound bed. Forexample, and not by way of limitation, Tβ4 or a Tβ4 isoform alone or incombination can be added in combination with any one or more of thefollowing agents: VEGF, KGF, FGF, PDGF, TGFβ, IGF-1, IGF-2, IL-1,prothymosin α and thymosin α1 in a wound-healing effective amount.

In another aspect, the invention is useful for repair of tissueresulting from injuries due to surgical procedures, irradiation,laceration, toxic chemicals, viral infections, bacterial infections orburns. Additionally, the invention is useful for revitalizing scartissue resulting from any number of procedures, accidents or trauma. Theterm “scar tissue” means fibrotic or collagenous tissue formed duringthe healing of a wound or other morbid process. For example, Tβ4 can beincluded in a controlled release matrix which can be positioned inproximity to damaged tissue thereby promoting regeneration, repairand/or revascularization of such tissue. The term “controlled releasematrix” means any composition that allows for the release of a bioactivesubstance which is mixed or admixed therein. The matrix can be a solidcomposition, a porous material (such as a scaffold, mesh, or sponge), ora semi-solid, gel or liquid suspension containing bioactive substances.The term “bioactive material” means any composition that modulatestissue repair when used in accordance with the method of the presentinvention. The bioactive materials or matrix can be introduced by meansof injection, surgery, catheters or any other means suitable formodulating tissue repair.

It is envisioned that the methods and compositions of the invention canbe used to aid wound healing and repair in guided tissue regeneration(GTR) procedures. Such procedures are currently used by those skilled inthe medical arts to accelerate wound healing. Typically, nonresorbableor bioabsorbable materials are used to accelerate wound healing bypromoting the repopulation of the wound area with cells which form thearchitectural and structural matrix of the tissue. For example, themethods and compositions of the invention can be used in aiding tissuerepair or regeneration at an ulcer site in a human or other subject byplacing a composition containing a bioreasorbable polymer and Tβ4 at thesite in need of tissue repair or regeneration such that the compositionis effective for aiding tissue regeneration by releasing a wound-healingeffective amount of Tβ4 at the site.

In another aspect, the invention is useful for the purposes of promotingtissue growth during the process of tissue engineering. As used herein,“tissue engineering” is defined as the creation, design, and fabricationof biological prosthetic devices, in combination with synthetic ornatural materials, for the creation, augmentation or replacement of bodytissues and organs. Thus, the present method can be used to augment thedesign and growth of human tissues outside the body, for laterimplantation inside the body, or augment the design and growth of atissue inside the body to repair or replace diseased or damaged tissue.For example, Tβ4 may be useful in promoting the growth of skin graftreplacements which are used as a therapy in the treatment of burns andulcers.

In another aspect of tissue engineering, Tβ4 can be included in externalor internal devices containing human tissue designed to replace thefunction of a diseased internal tissue. This approach involves isolatingcells from the body, placing them on or within three-dimensionalmatrices and implanting the new system inside the body or using thesystem outside the body. The methods and compositions of the inventioncan be used and included in such matrices to promote the growth oftissues contained in the matrices. For example, Tβ4 can be included in atissue engineered construct to promote the growth of the cells containedin the construct. It is envisioned that the method of the invention canbe used to augment tissue repair, regeneration and engineering inendothelial cell-related products which may contain cartilage,cartilage-bone composites, bone, central nervous system tissues, muscle,liver, pancreatic islet (insulin-producing) cells, urogenital tissues,breast and tissues for gene therapy applications.

The present invention further provides methods and compositions formodulating female reproductive tract function. Growth factors have beenshown to play a role in cyclic mitosis and differentiation ofendometrial cellular components, recruitment of macrophages indecidualizing the endometrium, endometrial-trophoblast interactions,early pregnancy maintenance, and endometrial functional regeneration.The term “modulate” as used herein, denotes a modification of anexisting condition or biologic state. Modulation of a condition asdefined herein, encompasses both an increase or a decrease in thedeterminants affecting the existing condition. For example,administration of Tβ4 could be used to augment uterine functions in acondition where the promotion of endothelial cell growth is desired. Forexample, the uterus may be treated with Tβ4 to promote the growth anddevelopment of placental membranes or endometrial growth or the repairof these tissue following tissue injury. Furthermore, treatment with Tβ4may be used to promote and maintain a pregnancy by facilitatingendometrial-trophoblast interaction. Alternatively, antagonist to Tβ4could be administered to modulate conditions of excessive endometrialgrowth in which the level of Tβ4 is excessive in comparison to a normalbiological condition. In addition, Tβ4 in combination with other agents,such as thymosin α1, may be desirable for treating disorders of thereproductive tract.

The therapeutic approaches described herein involve various routes ofadministration or delivery of reagents or compositions comprising theTβ4 of the invention including any conventional administrationtechniques (for example, but not limited to, topical administration,local injection, inhalation, or systemic administration), to a subjectwith a wound or tissue in need of healing or repair. Administration ofTβ4, as described above, can accelerate wound healing, increase cellmigration into a wound site, induce the formation of tissue repair orregeneration, or promote the growth and development of the endometrium.The reagent, formulation or composition may also be targeted to specificcells or receptors by any method described herein or by any method knownin the art of delivering, targeting Tβ4 polypeptides and expressinggenes encoding Tβ4. For example, the methods and compositions using orcontaining Tβ4 of the invention may be formulated into pharmaceuticalcompositions by admixture with pharmaceutically acceptable non-toxicexcipients or carriers. Such compositions may be prepared for parenteraladministration, particularly in the form of liquid solutions orsuspensions in aqueous physiological buffer solutions; for oraladministration, particularly in the form of tablets or capsules; or forintranasal administration, particularly in the form of powders, nasaldrops, or aerosols. Sustained release compositions are also encompassedby the present invention. Compositions for other routes ofadministration may be prepared as desired using standard methods.

A composition of the invention containing Tβ4 may be convenientlyadministered in unit dosage form, and may be prepared by any of themethods well known in the pharmaceutical art, for example as describedin Remington's Pharmaceutical Sciences (Mack Pub. Co., Easton, Pa.,1990). Formulations for parenteral administration may contain as commonexcipients sterile water or saline, polyalkylene glycols such aspolyethylene glycol, oils of vegetable origin, hydrogenatednaphthalenes, and the like. In particular, biocompatible, biodegradablelactide polymer, lactide/glycolide copolymer, orpolyoxethylene-polyoxypropylene copolymers are examples of excipientsfor controlling the release of a compound of the invention in vivo.Other suitable parenteral delivery systems include ethylene-vinylacetate copolymer particles, osmotic pumps, implantable infusionsystems, and liposomes. Formulations for inhalation administration maycontain excipients such as lactose, if desired. Inhalation formulationsmay be aqueous solutions containing, for example, polyoxyethylene-9-1auryl ether, glycocholate and deoxycholate, or they may be oilysolutions for administration in the form of nasal drops. If desired, thecompounds can be formulated as a gel to be applied intranasally.Formulations for parenteral administration may also include glycocholatefor buccal administration.

The composition of the liposome is usually a combination ofphospholipids, particularly high-phase-transition-temperaturephospholipids, usually in combination with steroids, especiallycholesterol. Other phospholipids or other lipids may also be used. Thephysical characteristics of liposomes depend on pH, ionic strength, andthe presence of divalent cations.

Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, sphingolipids,cerebrosides, and gangliosides. Particularly useful arediacylphosphatidylglycerols, where the lipid moiety contains from 14-18carbon atoms, particularly from 16-18 carbon atoms, and is saturated.Illustrative phospholipids include egg phosphatidylcholine,dipalmitoylphosphatidylcholine and distearoylphosphatidyl-choline.

The targeting of liposomes has been classified based on anatomical andmechanistic factors. Anatomical classification is based on the level ofselectivity, for example, organ-specific, cell-specific, andorganelle-specific. Mechanistic targeting can be distinguished basedupon whether it is passive or active. Passive targeting utilizes thenatural tendency of liposomes to distribute to cells of thereticulo-endothelial system (RES) in organs which contain sinusoidalcapillaries. Active targeting, on the other hand, involves alteration ofthe liposome by coupling the liposome to a specific ligand such as amonoclonal antibody, sugar, glycolipid, or protein, or by changing thecomposition or size of the liposome in order to achieve targeting toorgans and cell types other than the naturally occurring sites oflocalization.

The surface of the targeted delivery system may be modified in a varietyof ways. In the case of a liposomal targeted delivery system, lipidgroups can be incorporated into the lipid bilayer of the liposome inorder to maintain the targeting ligand in stable association with theliposomal bilayer. Various linking groups can be used for joining thelipid chains to the targeting ligand. In general, the compounds bound tothe surface of the targeted delivery system will be ligands andreceptors which will allow the targeted delivery system to find and“home in” on the desired cells. A ligand may be any compound of interestwhich will bind to another compound, such as a receptor.

The therapeutic agents useful in the method of the invention can beadministered parenterally by injection or by gradual perfusion overtime. Administration may be intravenously, intraperitoneally,intrarmuscularly, subcutaneously, intracavity, or transdermally.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's intravenousvehicles include fluid and nutrient replenishers, electrolytereplenishers (such as those based on Ringer's dextrose), and the like.Preservatives and other additives may also be present such as, forexample, antimicrobials, anti-oxidants, chelating agents and inert gasesand the like.

The invention also includes a pharmaceutical composition comprising aherapeutically effective amount of Tβ4 or a Tβ4 isoform in apharmaceutically cceptable carrier. Such carriers include those listedabove with reference to arenteral administration.

The actual dosage or reagent, formulation or composition that modulatesa issue repair process, fibrotic disorder, a sclerotic disorder, a cellproliferative isorder, or wound healing depends on many factors,including the size and health of subject. However, one of ordinary skillin the art can use the following teachings describing the methods andtechniques for determining clinical dosages (Spilker B., Guide toClinical Studies and Developing Protocols, Raven Press Books, Ltd., NewYork, 1984, pp. 7-13, 54-60; Spilker B., Guide to Clinical Trials, RavenPress, Ltd., New York, 1991, pp. 93-101; Craig C., and R. Stitzel, eds.,Modern Pharmacology, 2d ed., Little, Brown and Co., Boston, 1986, pp.127-33; T. Speight, ed., Avery's Drug Treatment: Principles and Practiceof Clinical Pharmacology and Therapeutics, 3d ed., Williams and Wilkins,Baltimore, 1987, pp. 50-56; R. Tallarida, R. Raffa and P. McGonigle,Principles in General Pharmacology, Springer-Verlag, N.Y., 1988, pp.18-20) or to determine the appropriate dosage to use.

Antibodies that Bind to Tβ4

Antibodies to Tβ4 peptide or fragments could be valuable as diagnostictools to aid in the detection of diseases in which Tβ4 is a pathologicalfactor. Further, use of antibodies which bind to Tβ4 and inhibit orprevent the actions of Tβ4 are included in the present invention.Therapeutically, antibodies or fragments of the antibody molecule couldalso be used to neutralize the biological activity of Tβ4 in diseaseswhere Tβ4 is over expressed. Such antibodies can recognize an epitope ofTβ4 or fragments thereof suitable for antibody recognition andneutralization of Tβ4 activity. As used in this invention, the term“epitope” refers to an antigenic determinant on an antigen, such as aTβ4 peptide, to which the paratope of an antibody, such as anTβ4-specific antibody, binds. Antigenic determinants usually consist ofchemically active surface groupings of molecules, such as amino acids orsugar side chains, and can have specific three-dimensional structuralcharacteristics, as well as specific charge characteristics.

Preparation of an antibody requires a substantially purified moiety thatcan provide an antigenic determinant. The term “substantially pure” asused herein refers to Tβ4, or variants thereof, which is substantiallyfree of other proteins, lipids, carbohydrates or other materials withwhich it is naturally associated. Substantially purified or “isolated”refers to molecules, either nucleic or amino acid sequences, that areremoved from their natural environment, isolated or separated, and areat least 60% free, preferably 75% free, and most preferably 90% freefrom other components with which they are naturally associated. Oneskilled in the art can isolate Tβ4 or a Tβ4 isoform using standardtechniques for protein purification. The substantially pure peptide willyield a single major band on a non-reducing polyacrylamide gel. Thepurity of the Tβ4 peptide can also be determined by amino-terminal aminoacid sequence analysis. Tβ34 or a Tβ4 isoform peptide includesfunctional fragments of the peptide, as long as the activity of Tβ4 or aTβ4 isoform remains. Smaller peptides containing the biological activityof Tβ4 or a Tβ4 isoform are included in the invention. As used in thepresent invention, the term “antibody” includes, in addition toconventional antibodies, such protein fragments that have the ability torecognize specifically and bind the Tβ4 protein or variants thereof.Regions of the gene that differ at the protein level are well defined. Aprotein can be raised by expression of the wild type (wt) gene or of thevariants, or, preferably, fractions therefore. For example, the nucleicacid sequence can be cloned into expression vectors. According to thisembodiment, the sequence of interest can first be obtained by employingPCR, as described above, or from a synthetic gene construction withoverlapping and ligated synthetic oligonucleotides. Another alternativewould involve synthesis of a short peptide. All those methodologies arewell known to one skilled in the art. See, for example, Ausubel et al.,CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Volumes 1 and 2 (1987), withsupplements, and Maniatis et al., MOLECULAR CLONING, A LABORATORYMANUAL, Cold Spring Harbor Laboratory, all of which are incorporatedherein by reference.

The invention provides a method for detecting Tβ4, or variants thereof,which includes contacting an anti-Tβ4 antibody with a sample suspectedof containing Tβ4, (e.g., cell or protein) and detecting binding to theantibody. An antibody which binds to Tβ4 peptide is labeled with acompound which allows detection of binding to Tβ4. There are manydifferent labels and methods of labeling known to those of ordinaryskill in the art. Examples of the types of labels which can be used inthe present invention include enzymes, radioisotopes, fluorescentcompounds, colloidal metals, chemiluminescent compounds, phosphorescentcompounds, and bioluminescent compounds. Those of ordinary skill in theart will know of other suitable labels for binding to the antibody, orwill be able to ascertain such, using routine experimentation. Forpurposes of the invention, an antibody specific for Tβ4 peptide may beused to detect the level of Tβ4 in biological fluids and tissues. Anyspecimen containing a detectable amount of antigen can be used. Thelevel of Tβ4 in the suspect cell can be compared with the level in anormal cell to determine whether the subject is predisposed to a Tβ4associated increase in angiogenesis or wound healing.

Use of antibodies for the diagnostic methods of the invention includes,for example, immunoassays in which the antibodies can be utilized inliquid phase or bound to a solid phase carrier. In addition, theantibodies in these immunoassays can be detectably labeled in variousways. Examples of types of immunoassays which can utilize antibodies ofthe invention are competitive and non-competitive immunoassays in eithera direct or indirect format. Examples of such immuno assays are theradioimmunoassay (RIA) and the sandwich (immunometric) assay. Detectionof the antigens using the antibodies of the invention can be doneutilizing immunoassays which are run in either the forward, reverse, orsimultaneous modes, including immunohistochemical assays onphysiological samples. Those of skill in the art will know, or canreadily discern, other immunoassay formats without undueexperimentation.

Tβ4 antibodies can be bound to many different carriers and used todetect the presence of an antigen comprising the peptide of theinvention. Examples of well-known carriers include glass, polystyrene,polypropylene, polyethylene, dextran, nylon, amylases, natural andmodified celluloses, polyacrylamides, agaroses and magnetite. The natureof the carrier can be either soluble or insoluble for purposes of theinvention. Those skilled in the art will know of other suitable carriersfor binding antibodies, or will be able to ascertain such, using routineexperimentation.

Another technique which may also result in greater sensitivity consistsof coupling the antibodies to low molecular weight haptens. Thesehaptens can then be specifically detected by means of a second reaction.For example, it is common to use such haptens as biotin, which reactswith avidin, or dinitrophenyl, puridoxal, and fluorescein, which canreact with specific antihapten antibodies.

The invention includes use of antibodies immunoreactive with Tβ4 peptideor functional fragments thereof. Antibody which consists essentially ofpooled monoclonal antibodies with different epitopic specificities, aswell as distinct monoclonal antibody preparations are provided.Monoclonal antibodies are made from antigen containing fragments of theprotein by methods well known to those skilled in the art (Kohler, etal., Nature, 256:495, 1975). The term antibody as used in this inventionis meant to include intact molecules as well as fragments thereof, suchas Fab and F(ab′)₂, Fv and SCA fragments which are capable of binding anepitopic determinant on Tβ4.

(1) An Fab fragment consists of a monovalent antigen-binding fragment ofan antibody molecule, and can be produced by digestion of a wholeantibody molecule with the enzyme papain, to yield a fragment consistingof an intact light chain and a portion of a heavy chain.

(2) An Fab′ fragment of an antibody molecule can be obtained by treatinga whole antibody molecule with pepsin, followed by reduction, to yield amolecule consisting of an intact light chain and a portion of a heavychain. Two Fab′ fragments are obtained per antibody molecule treated inthis manner.

(3) An (Fab′)₂ fragment of an antibody can be obtained by treating awhole antibody molecule with the enzyme pepsin, without subsequentreduction. A (Fab′)₂ fragment is a dimer of two Fab′ fragments, heldtogether by two disulfide bonds.

(4) An Fv fragment is defined as a genetically engineered fragmentcontaining the variable region of a light chain and the variable regionof a heavy chain expressed as two chains.

(5) A single chain antibody (“SCA”) is a genetically engineered singlechain molecule containing the variable region of a light chain and thevariable region of a heavy chain, linked by a suitable, flexiblepolypeptide linker.

Alternatively, a therapeutically or diagnostically useful anti-Tβ4antibody may be derived from a “humanized” monoclonal antibody.Humanized monoclonal antibodies are produced by transferring mousecomplementary determining regions from heavy and light variable chainsof the mouse immunoglobulin into a human variable domain, and thensubstituting human residues in the framework regions of the murinecounterparts. The use of antibody components derived from humanizedmonoclonal antibodies obviates potential problems associated with theimmunogenicity of murine constant regions. General techniques forcloning murine immunoglobulin variable domains are described, forexample, by Orlandi et al., Proc. Natl. Acad. Sci. USA 86: 3833 (1989),which is hereby incorporated in its entirety by reference. Techniquesfor producing humanized monoclonal antibodies are described, forexample, by Jones et al., Nature 321: 522 (1986); Riechmann et al.,Nature 332: 323 (1988); Verhoeyen et al., Science 239: 1534 (1988);Carter et al., Proc. Nat'l Acad. Sci. USA 89: 4285 (1992); Sandhu, Crit.Rev. Biotech. 12: 437 (1992); and Singer et al., J. Immunol. 150: 2844(1993), which are hereby incorporated by reference.

Antibodies of the invention also may be derived from human antibodyfragments isolated from a combinatorial immunoglobulin library. See, forexample, Barbas et al., METHODS: A COMPANION TO METHODS IN ENZYMOLOGY,VOL. 2, page 119 (1991); Winter et al., Ann. Rev. Immunol. 12: 433(1994), which are hereby incorporated by reference. Cloning andexpression vectors that are useful for producing a human immunoglobulinphage library can be obtained, for example, from STRATAGENE CloningSystems (La Jolla, Calif.).

Methods and Compositions for Treating or Diagnosing Tβ4-AssociatedDisorders

In another embodiment of the invention, a method of diagnosing apathological state in a subject suspected of having a pathologycharacterized by a disorder associated with Tβ4 is provided. The methodincludes obtaining a sample suspected of containing Tβ4 from thesubject, determining the level of Tβ4 in the sample and comparing thelevel of Tβ4 in the sample to the level of Tβ4 in a normal standardsample. Such conditions include, but are not limited to subjects havingcell proliferative disorders, recurrent wounds, tissue repair disorders,fibrotic tissue disorders, chronic ulcers and other disorders describedherein. Such disorders further include those associated with the variousTβ4 isoforms, known or not yet identified.

The term “cell-proliferative disorder” denotes malignant as well asnon-malignant cell populations which often appear to differ from thesurrounding tissue both morphologically and genotypically. Malignantcells (i.e. cancer) develop as a result of a multistep process. Suchdisorders may be detected using the methods of the current invention.For example, a sample suspected of containing Tβ4 is obtained from asubject, the level of Tβ4 peptide is determined and compared with thelevel of Tβ4 peptide in a normal tissue sample. The level of Tβ4 can bedetermined by any number of methods including, for example, immunoassayusing anti-Tβ4 peptide antibodies. Other variations of such assaysinclude radioimmunoassay (RIA), ELISA and immunofluorescence.Alternatively, nucleic acid probes can be used to detect and quantifyTβ4 peptide mRNA for the same purpose. Such detection methods arestandard in the art.

In another embodiment, the invention provides a method for amelioratinga wound healing disorder associated with Tβ4 or a Tβ4 isoform, includingtreating a subject having the disorder with a composition that regulatesTβ4 activity. The term “ameliorate” denotes a lessening of thedetrimental effect of the disease-inducing response in the subjectreceiving therapy. Where the disease is due to an abnormally high levelof Tβ4, the administration of an agent, such as an antagonist of Tβ4activity, may be effective in decreasing the amount of Tβ4 activity.Alternatively, where the disease is due to an abnormally low level ofTβ4, the administration of Tβ4 or an agent that increases Tβ4 activity,such as an agonist, may be effective in increasing the amount of Tβ4activity.

In yet another embodiment, the invention provides a method of treating asubject having a wound healing disorder characterized by recurrent orslow to heal wounds or wounds that are chronic non-healing woundsassociated with altered Tβ4 or Tβ4 isoform gene expression in a subject.The method includes administering to a subject having the disorder awound-healing effective amount of an agent which modulates Tβ4 geneexpression, thereby treating the disorder. The term “modulate” refers toinhibition or suppression of Tβ4 expression when Tβ4 is over expressed,and induction of expression when Tβ4 is under expressed. The term“wound-healing effective amount” means that amount of Tβ4 agent which iseffective in modulating Tβ4 gene expression resulting in reducing thesymptoms of the Tβ4 associated wound healing disorder.

An agent which modulates Tβ4 or Tβ4 isoform gene expression may be apolynucleotide for example. The polynucleotide may be an antisense, atriplex agent, or a ribozyme. For example, an antisense directed to thestructural gene region or to the promoter region of Tβ4 may be utilized.

When a wound healing disorder is associated with the expression of Tβ4,a therapeutic approach which directly interferes with the translation ofTβ4 mRNA into protein is possible. For example, an antisense nucleicacid or a ribozyme can be used to bind to the Tβ4 RNA or to cleave it.Antisense RNA or DNA molecules bind specifically with a targeted gene'sRNA message, interrupting the expression of that gene's protein product.The antisense binds to the mRNA, forming a double stranded moleculewhich cannot be translated by the cell. Antisense oligonucleotides ofabout 15-25 nucleotides are preferred since they are easily synthesizedand have an inhibitory effect just like antisense RNA molecules. Inaddition, chemically reactive groups, such as iron-linkedethylenediaminetetraacetic acid FDTA-Fe) can be attached to an antisenseoligonucleotide, causing cleavage of the RNA at the site ofhybridization. These and other uses of antisense methods to inhibit thein vitro translation of genes are well known in the art (Marcus-Sakura,Anal., Biochem., 172:289, 1988).

Antisense nucleic acids are DNA or RNA molecules that are complementaryto at least a portion of a specific mRNA molecule (Weintraub, ScientificAmerican, 262; 40, 1990). In the cell, the antisense nucleic acidshybridize to the corresponding mRNA, forming a double-stranded molecule.The antisense nucleic acids interfere with the translation of the mRNA,since the cell will not translate a mRNA that is double-stranded.Antisense oligomers of about 15 nucleotides are preferred, since theyare easily synthesized and are less likely to cause problems than largermolecules when introduced into the target Tβ4 producing cell. The use ofantisense methods to inhibit the in vitro translation of genes is wellknown in the art (Marcus-Sakura, Anal. Biochem., 172:289, 1988).

Use of an oligonucleotide to stall transcription is known as the triplexstrategy since the oligomer winds around double-helical DNA, forming athree-strand helix. Therefore, these triplex compounds can be designedto recognize a unique site on a chosen gene (Maher, et al., AntisenseRes. and Dev., 1(3):227, 1991; Helene, C., Anticancer Drug Design,6(6):569, 1991).

Ribozymes are RNA molecules possessing the ability to specificallycleave other single-stranded RNA in a manner analogous to DNArestriction endonucleases. Through the modification of nucleotidesequences which encode these RNAs, it is possible to engineer moleculesthat recognize specific nucleotide sequences in an RNA molecule andcleave them (Cech, J. Amer. Med. Assn., 260: 3030, 1988). A majoradvantage of this approach is that, because they are sequence-specific,only mRNAs with particular sequences are inactivated.

There are two basic types of ribozymes namely, tetrahymena-type(Hasselhoff; Nature, 334:585, 1988) and “hammerhea”-type.Tetrahymena-type ribozymes recognize sequences which are four bases inlength, while “hammerhead”-type ribozymes recognize base sequences 11-18bases in length. The longer the recognition sequence, the greater thelikelihood that the sequence will occur exclusively in the target mRNAspecies.

These and other uses of antisense methods to inhibit the in vivotranslation of genes are well known in the art (e.g., De Mesmaeker, etal., 1995. Backbone modifications in oligonucleotides and peptidenucleic acid systems. Curr. Opin. Struct. Biol. 5:343-355; Gewirtz, A.M., et al., 1996b. Facilitating delivery of antisenseoligodeoxynucleotides: Helping antisense deliver on its promise; Proc.Natl. Acad. Sci. U.S.A. 93:3161-3163; Stein, C. A. A discussion ofG-tetrads 1996. Exploiting the potential of antisense: beyondphosphorothioate oligodeoxynucleotides. Chem. and Biol. 3:319-323).

Delivery of antisense, triplex agents, ribozymes, competitive inhibitorsand the like can be achieved using a recombinant expression vector suchas a chimeric virus or a colloidal dispersion system. Various viralvectors which can be utilized for gene therapy as taught herein includeadenovirus, herpes virus, vaccinia, or, preferably, an RNA virus such asa retrovirus. Preferably, the retroviral vector is a derivative of amurine or avian retrovirus. Examples of retroviral vectors in which asingle foreign gene can be inserted include, but are not limited to:Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus(HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus(RSV). A number of additional retroviral vectors can incorporatemultiple genes. All of these vectors can transfer or incorporate a genefor a selectable marker so that transduced cells can be identified andgenerated. By inserting a polynucleotide sequence of interest into theviral vector, along with another gene which encodes the ligand for areceptor on a specific target cell, for example, the vector is nowtarget specific. Retroviral vectors can be made target specific byinserting, for example, a polynucleotide encoding a sugar, a glycolipid,or a protein. Preferred targeting is accomplished by using an antibodyto target the retroviral vector. Those of skill in the art will know of,or can readily ascertain without undue experimentation, specificpolynucleotide sequences which can be inserted into the retroviralgenome to allow target specific delivery of the retroviral vectorcontaining the antisense polynucleotide.

Since recombinant retroviruses are defective, they require assistance inorder to produce infectious vector particles. This assistance can beprovided, for example, by using helper cell lines that contain plasmidsencoding all of the structural genes of the retrovirus under the controlof regulatory sequences within the LTR. These plasmids are missing anucleotide sequence which enables the packaging mechanism to recognizean RNA transcript for encapsidation. Helper cell lines which havedeletions of the packaging signal include but are not limited to Ψ2,PA317 and PA12, for example. These cell lines produce empty virions,since no genome is packaged. If a retroviral vector is introduced intosuch cells in which the packaging signal is intact, but the structuralgenes are replaced by other genes of interest, the vector can bepackaged and vector virion produced.

Alternatively, NIH 3T3 or other tissue culture cells can be directlytransfected with plasmids encoding the retroviral structural genes gag,pol and env, by conventional calcium phosphate transfection. These cellsare then transfected with the vector plasmid containing the genes ofinterest. The resulting cells release the retroviral vector into theculture medium.

A targeted delivery system for delivery of nucleic acids as describedherein includes a colloidal dispersion system. Colloidal dispersionsystems include macromolecule complexes, nanocapsules, microspheres,beads, gene activated matrices and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Thepreferred colloidal system of this invention is a liposome. Liposomesare artificial membrane vesicles which are useful as delivery vehiclesin vitro and in vivo. It has been shown that large unilamellar vesicles(LUV), which range in size from 0.2-4.0 μm can encapsulate a substantialpercentage of an aqueous buffer containing large macromolecules. RNA,DNA and intact virions can be encapsulated within the aqueous interiorand be delivered to cells in a biologically active form (Fraley, et al.,Trends Biochem. Sci., 6:77, 1981). In addition to mammalian cells,liposomes have been used for delivery of polynucleotides in plant, yeastand bacterial cells. In order for a liposome to be an efficient genetransfer vehicle, the following characteristics should be present: (1)encapsulation of the genes of interest at high efficiency while notcompromising their biological activity; (2) preferential and substantialbinding to a target cell in comparison to non-target cells; (3) deliveryof the aqueous contents of the vesicle to the target cell cytoplasm athigh efficiency; and (4) accurate and effective expression of geneticinformation (Mannino, et al., Biotechniques, 6:682, 1988).

Pathologically, Tβ4 may be involved in diseases in which there is anovergrowth of blood vessels, such as cancer, tumor formation and growth,diabetic retinopathy, neovascular glaucoma, rheumatoid arthritis andpsoriasis.

The ingrowth of capillaries and ancillary blood vessels is essential forgrowth of solid tumors and is thus an unwanted physiological responsewhich facilitates the spread of malignant tissue and metastases.Inhibition of angiogenesis and the resultant growth of capillaries andblood vessels is therefore a component of effective treatment ofmalignancy in use of treatment of cancer patients.

Thus, in another embodiment, the invention provides a method ofinhibiting angiogenesis in a subject, including administering to thesubject a composition containing an agent which regulates Tβ4 activity.The composition may include agents that regulate angiogenesis, forexample agents that affect thymosin α1, PDGF, VEGF, IGF, FGF and TGFβ.For example, the inhibition of angiogenesis and endothelial cellmigration can be beneficial in controlling the growth of solid tumors.Most, if not all solid tumors, like normal tissue, require a steady andsufficient blood supply for optimal growth. Tumors are known to make useof angiogenic growth factors to attract new blood vessels and ascertainsupply with sufficient amounts of nutrients to sustain their growth.Many tumors are well vascularized and the inhibition of the formation ofan adequate blood supply to the tumor by inhibition of tumorvascularization, as a result of inhibition of angiogenesis, isbeneficial in tumor growth control. Without a strong blood supply, rapidand prolonged growth of tumor tissue cannot be sustained. Thus, agentsthat inhibit Tβ4 activity may be used to prevent neoplastic growth. TheTβ4 inhibiting agent may be administered orally, parenterally,topically, intravenously, or systemically. In addition, for inhibitingtumor cell proliferation and tumor growth, the agent may be administeredlocally directly to the tumor or as a part of a deposited slow releaseformulation. Administration may be on a daily basis for as long asneeded to inhibit angiogenesis, endothelial cell proliferation, tumorcell proliferation or tumor growth. Alternatively, a slow releaseformulation may continue for as long as needed to control tumor growth.This dosage regimen may be adjusted to provide the optimum therapeuticresponse. For example, several divided doses may be administered dailyor the dose may be proportionally reduced as indicated by the exigenciesof the therapeutic situation.

In this regard, the compositions of this invention that are useful asinhibitors of angiogenesis, endothelial cell proliferation, tumor cellproliferation and tumor growth contain a pharmaceutically acceptablecarrier and an amount of Tβ4 modulating agent effective to inhibit tumoror endothelial cell proliferation. Such compositions may also includepreservatives, antioxidants, immunosuppressants and other biologicallyand pharmaceutically effective agents which do have effects on tumorgrowth but which do not exert a detrimental effect on the Tβ4 modulatingagent. For treatment of tumor cells the composition may include achemotherapeutic agent, for example an anti-cancer agent whichselectively kills the faster replicating tumor cells, many of which areknown and clinically used. Exemplary anticancer agents includeinephalan, cyclophosphamide, methotrexate, adriamycin and bleomycin.

Screen for Compounds which Modulate Tβ4 Activity

In another embodiment, the invention provides a method for identifying acompound that modulates Tβ4 activity, angiogenesis activity or woundhealing activity. The method includes incubating components includingthe compound and Tβ4 under conditions sufficient to allow the componentsto interact and determining the effect of the compound on Tβ4 activitybefore and after incubating in the presence of the compound. Compoundsthat affect Tβ4 activity (e.g., antagonists and agonists) includepeptides, peptidomimetics, polypeptides, chemical compounds, mineralssuch as zincs, and biological agents. Tβ4 activity can be assayed usingthe methodology as described in the present Examples.

The present Examples are meant to illustrate, but not limit the scope ofthe appended claims. Accordingly, one skilled in the art will recognizea number of equivalent materials and methods, which are intend to becovered by the present invention and disclosure.

Example 1 In Vivo Wound Healing is Accelerated by Tβ4

Tβ4, whether administered topically or intraperitoneally, significantlyaccelerated wound healing as compared to untreated wounds (FIGS. 2 and3). Full thickness 8 mm punch biopsy wounds were made on the dorsalsurface of rats as previously reported (Bhartiya et al., J. Cell.Physiol. 150:312, 1992; Sihhu et al., J. Cell, Physiol. 169:108, 1996)and Tβ4 was given topically at the time of wounding (5 μg in 50 μl) andagain after 48 hours. Controls for the topical treatment receivedidentical amounts of saline at the time of wounding and at 48 hours.Additional rats received intraperitoneal injections at the time ofwounding (60 μg in 300 μl) and again every other day (e.g., days 0, 2,4, and 6). Controls for these animals received identical amounts ofsaline intra-peritoneally on the same injection schedule. On days 4 and7 post-wounding, measurements were made of the wound size. At days 8 and9 post-wounding, tissue was collected and fixed in 10% bufferedformalin. The samples were sectioned and stained with H&E and Masson'sTrichrome (American Histolabs, Gaithersburg, Md.).

Histological sections were used to measure the re-epithelialization andthe contraction of the wound using an ocular micrometer. Epidermalmigration was determined by measuring the lengths of the tongues ofepithelium migrating form either side of the wound over the wound bedfrom the zone of proliferation at the margin of the uninjured andwounded skin. Epidermal thickness was also measured beginning at thejunction of the uninjured and proliferating epidermis. The thickness wasmeasured vertically from the basement membrane to the most superficiallayer of the migrating epidermis at every 200 microns. The meanepidermal thickness of each migrating tongue of epidermis was thencomputed from each wound. Vessel counts were performed by firstidentifying vascular spaces by their endothelial lining. All suchvessels in the wound bed were counted including those at the junction ofthe dermis and the subcutis, since angiogenesis into the wounds occursto a great extent from these vessels. The numbers were averaged intovessel counts per 10 high powered fields (40×).

The effect of Tβ4 on wound healing was studied in a full thicknesscutaneous rat wound model. FIG. 1 shows a diagram of the wound site thatextends form the epidermis to the fat/muscle layer. This model allowedmeasurement of two parameters: the re-epithelialization (gap) and thecontraction (width) of the wound. Wounds treated topically with Tβ4showed about a 15% decrease in width and about 15% decrease in gap inthe treated versus controls (FIGS. 2 and 3, respectively).

FIG. 2 shows a 15% decrease in wound width as compared to the salinecontrols as early as 4 days after wounding and continued until day 7.Intraperitoneal injection of Tβ4 resulted in a 18% decrease in woundwidth relative to saline treated controls at day 4 and 11% decrease atday 7. This trend was observed on the 4th day post wounding andcontinued through day 7 (*P≦0.0001, **P≦0.08, significant differencefrom media alone, student's t-test). These data demonstrate that Tβ4,when given either topically or systemically, increases woundre-epithelialization and contraction. Both topical and systemictreatment are equally effective. Lower doses of Tβ4 were testedincluding 2.5 μg and 0.5 μg in 50 μl for topical and 30 μg and 6 μg in300 μl for intraperitoneal injection but reduced or no effect,respectively, was observed on wound healing.

FIG. 3 shows an 18% decease in gap length as compared to saline controlswhen Tβ4 is administered topically, as early as 4 days after wounding.This trend continued to termination at day 7 (*P≦0.04, student'st-test). Intraperitoneal injections resulted in a 42% decrease in gapsize relative to saline treated controls. This decrease was observed onthe 4th day post wounding and continued through day 7 (**P≦0.0007,student's t-test). The increase in re-epithelialization was observed inwounds treated for 7 days and the rate of gap closure was slightlyaccelerated over that observed at day 4. A 62% decrease in gap size wasobserved in the Tβ4-treated wounds. Quantitation of epidermal migrationshowed a statistically significant 1.5 fold increase in migration ofepidermal tongues over the wound bed after topical treatment (Table 1).Quantitation of epithelial migration in intraperitoneally treated woundsshowed a statistically significant increase in migration of epidermaltongues as compared to controls (Table 1). There was no difference inthe thickness of the migrating epidermis between either of the Tβ4treatments and the control (Table 1). Histological sections of thewounds clearly show increased re-epithelialization in the treated woundsas compared to controls in 7 day wounds (FIG. 4).

TABLE 1 Morphometric Measurements of Control and Thymosin β4 TreatedSamples Parameter Control I.P. Topical Epidermal Migration 2403.3 ± 9.73168.3 ± 38.4* 3668.7 ± 56.6* (μm) Epidermal Thickness  128.2 ± 19.3 135.0 ± 11.7  142.3 ± 19.8 (μm) Vessels/10 HPF 1364.0 ± 15.0 2415.0 ±24.3* 2186.0 ± 11.8* HPF: high power field. *P ≦ 0.00001 by Welch'st-test, significantly different than control.

FIG. 4 shows a comparison of typical control (D) and Tβ4-treated (E andF) sections of 7 day wounds. Treatment with Tβ4 resulted in considerablecapillary ingrowth (FIGS. 4E and F, arrows). Vessel counts showed asignificant (about 2 fold) increase in the number of vessels in Tβ4treated wounds (Table 1). No increases in the number of macrophages inthe wounds were observed. There was no apparent increase in theaccumulation/biosynthesis of collagen in treated-Tβ4 wounds (FIGS. 5Band C vs A) supporting a decreased wound width and supporting a role forTβ4 in wound contraction. Both the topical and systemically treatedwound appeared similar although the wound contraction proceeded slightlymore quickly with the topical treatment.

Reduction of the wound size was observed in both experimental groups ascompared to control groups (FIGS. 2-4). More and larger blood vesselswere noted in the experimental groups as compared to the controls (FIG.4). Additionally, an increase in the accumulation/biosynthesis ofcollagen by Tβ4 treated wounds as compared to control suggests a rolefor Tβ4 in wound contraction and extracellular matrix deposition.Histological staining of these wounds demonstrated an increase incollagen density and extracellular matrix deposition when compared tocontrols. (FIG. 5).

Example 2 Migration Assays of Keratinocytes

Primary keratinocytes were prepared from either Balb/c or CD-1 newbornmice as described previously (Dlugosz et al., 1995). Cells were platedin calcium- and magnesium-free Eagle's Minimal Essential Medium (EMEM)containing 8% fetal calf serum treated with 8% Chelex (Bio-RadLaboratories, Hercules, Calif.), 20 units/ml penicillin-streptomycin,and the calcium concentration was adjusted to 0.25 mM. The followingday, cultures were washed with calcium- and magnesium-free phosphatebuffered saline, treated briefly with Trypsin (Life Technologies,Gaithersburg, Md.), washed with culture medium and resuspended in EMEMcontaining 0.05 mM calcium. Cells were used immediately in migrationassays.

Keratinocyte migration assays were carried out in Boyden chamber using12 μm pore polyester membranes (Poretics, Livermore, Calif.) coated witha 0.1 mg/ml solution of collagen IV in dH₂0 (Trevigen, Gaithersburg,Md.). Filters were then dried at least 1 h. Cells were harvested usingVersene or Trypsin (Life Technologies, Gaithersburg, Md.) andresuspended in Eagle's minimal essential medium with 0.05 mM Ca²⁺. Thebottom chamber was loaded with EMEM containing 0.01, 0.1, 10, 100, and1000 ng/ml of synthetic Tβ4. Conditioned medium from primary dermalfibroblasts and/or keratinocyte growth factor was added to several wellsas a positive control. Cells were added to the upper chamber at aconcentration of 50,000 cells per well. Chambers were incubated at 35C/7% CO₂ for 4-5 hours and the filters were then fixed and stained usingDiff-Quik (Baxter Healthcare Corporation, McGraw Park, Ill.). The cellsthat migrated through the filter were quantitated by counting the centerof each well at 10× using an Olympus CK2 microscope. Each condition wasassayed in triplicate wells and each experiment was repeated four timeswith different preparations of cells.

The results demonstrated that keratinocyte migrated in response to Tβ4after 4-5 hours of exposure. Migration was enhanced 2-3 fold (P≦0.003)over migration in the presence of media alone (FIG. 6) and at themaximal responding dose exceeded the positive control. The effect of Tβ4on migration, while showing slightly different dose curves depending onthe cell preparation and source, clearly showed a biphasic pattern with1000 ng/ml and 0.01 ng/ml showing the most migration and the middledoses showing less stimulation (but still greater than control media) inall 4 assays.

Example 3 Migration Assays of Corneal Epithelial Cells

Corneal Epithelial Cell migration assays were carried out in Boydenchamber using 12 μm pore polyester membranes (Poretics, Livermore,Calif.) coated with a 0.1 mg/ml solution of collagen IV in dH20(Trevigen, Gaithersburg, Md.). Filters were then dried at least 1 h.Cells were cultured and resuspended in Eagle's Minimal Essential Mediumwith 0.05 mM Ca²⁺. The bottom chamber was loaded with EMEM containing0.01, 0.1, 10, 100, and 1000 ng/ml of synthetic Tβ4. Conditioned mediumfrom primary dermal fibroblasts and/or keratinocyte growth factor wasadded to several wells as a positive control. Cells were added to theupper chamber at a concentration of 50,000 cells per well. Chambers wereincubated at 35 C/7% CO₂ for 4-5 hours and the filters were then fixedand stained using Diff-Quik (Baxter Healthcare Corporation, McGraw Park,Ill.). The cells that migrated through the filter were quantitated bycounting the center of each well at 10× using an Olympus CK2 microscope.Each condition was assayed in triplicate wells and each experiment wasrepeated four times with different preparations of cells. The resultsdemonstrated that corneal epithelial cell migrated in response to Tβ4after 4-5 hours of exposure. Migration was enhanced 2-3 fold overmigration in the presence of media alone (FIG. 7) with the highest levelof migration seen at 100 ng/ml of Tβ4.

Example 4 In Vivo Corneal Re-Epithelialization

To determine the effect of Tβ4 on corneal re-epithelialization in vivo,Rat corneas were de-epithelialized and treated with Tβ4. Filters weresoaked in heptanol, applied to the eye for 30 seconds, and then theepithelium was scraped. Various concentration of Tβ4 in saline wasapplied to the eye and at 24 hours the rats were sacrificed. The eyeswere fixed, sectioned and the degree of corneal epithelial migration (asmeasured in pixels) was determined using a microscope with an internalcaliper by a masked observer. The results demonstrate thatre-epithelialization of the cornea was increased 2-fold over untreatedcontrol in the presence of about 1 to 25 μg of Tβ4 (FIGS. 8 and 9). Inaddition, it was noted that Tβ4 treated eyes had reduced inflammationcompared to the non-treated corneas.

Example 5 Impaired Healing Model

Thymosin β4 also enhanced wound healing in an impaired model. Steroidtreatment reduces the rate of wound repair in mammals. Rats treated withsteroids such as hydrocortisone serve as a model of impaired woundhealing due to the delay observed in wound closure. Animals wereinjected intramuscularly every day with hydrocortisone. Steroid-treatedrats showed a significant increase in the level of healing when Tβ4 wasadded topically or injected intraperitoneally. At the initial timepoint, day 4, topically-treated animals showed little response (≦7% gapor width closure, N=3) compared to saline treatment. Intraperitonealinjection, however, resulted in a 28% decrease in gap size and a 14%decrease in wound width. At day 7, a response was observed with bothtopical treatment and intraperitoneal injection.

The gap in topically treated animals decreased by 39% compared to salinetreatment. The wound width decreased by 23%. Intraperitoneal injectionresulted in a 26% decrease in gap size and a 10% decease in wound width.Taken together, these demonstrate that Tβ4 is useful to treat chronic,as well as, acute wounds.

A number of embodiments of the present invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1. A composition for topical administration to skin, comprising a tissueregeneration promoting amount of human thymosin β4 of SEQ ID NO:3; humantransforming growth factor β1, and a pharmaceutically acceptable topicalvehicle.
 2. A composition for topical administration to skin comprisinga tissue regeneration promoting amount of human thymosin β4 of SEQ IDNO:3; human vascular endothelial growth factor and a pharmaceuticallyacceptable topical vehicle.
 3. The composition according to claim 2,wherein said vehicle is an oil in water, or water in oil emulsion.
 4. Amethod for improving damage to the skin, the method comprising: applyingtopically to said damaged skin a composition comprising a tissueregeneration promoting amount of human thymosin β4 of SEQ ID NO:3; and apharmaceutically acceptable topical vehicle.
 5. A method for improvingdamage to the skin, the method comprising: applying topically acomposition comprising a tissue regeneration promoting amount of humanthymosin β4 of SEQ ID NO:3, human transforming growth factor β1, and apharmaceutically acceptable topical vehicle.
 6. A method for improvingdamage to the skin, the method comprising: applying topically acomposition comprising a tissue regeneration promoting amount of humanthymosin β4 of SEQ ID NO:3, human vascular endothelial growth factor,and a pharmaceutically acceptable topical vehicle.
 7. The methodaccording to claim 6, wherein said skin damaged by irradiation orcontains scar tissue.
 8. The method according to claim 7, wherein saidcosmetically acceptable vehicle is an oil in water, or water in oilemulsion.
 9. A composition for topical treatment of skin, comprising athymosin beta 4 of SEQ ID NO:3 and a vehicle acceptable for topicaladministration, further comprising transforming growth factor beta,vascular endothelial growth factor, or both.
 10. The composition ofclaim 9, further comprising transforming growth factor beta.
 11. Thecomposition of claim 10, wherein said transforming growth factor beta istransforming growth factor beta
 1. 12. The composition of claim 9,further comprising vascular endothelial growth factor.
 13. Thecomposition of claim 9, wherein said vehicle is a lotion, cream,suspension, dispersion or oil.
 14. A method of treating damaged skintissue in a subject comprising topically administering the compositionof claim 9, to said subject.
 15. A method of regenerating orrevitalizing skin tissue in a subject comprising topically administeringthe composition of claim 9, to said subject.
 16. The method of claim 14,wherein said vehicle is a lotion, cream, suspension, dispersion or oil.17. The composition of claim 1 wherein said skin is in need of repair.18. The composition of claim 2 wherein said skin is in need of repair.19. The method of claim 4 wherein said skin is in need of repair. 20.The method of claim 5 wherein said skin is in need of repair.
 21. Themethod of claim 6 wherein said skin is in need of repair.